This invention relates generally to hypoxia-inducible DNA-binding proteins and more specifically to DNA binding proteins that are modified such that they are stable under non-hypoxic as well as hypoxic conditions.
Mammals require molecular oxygen (O2) for essential metabolic processes including oxidative phosphorylation in which O2 serves as electron acceptor during ATP formation. Systemic, local, and intracellular homeostatic responses elicited by hypoxia (the state in which O2 demand exceeds supply) include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann, Physiol. Rev. 72:449-489, 1992), neovascularization in ischemic myocardium (White et al., Circ. Res. 71:1490-1500, 1992), and glycolysis in cells cultured at reduced O2 tension (Wolfe et al., Eur. J. Biochem. 135:405-412, 1983). These adaptive responses either increase O2 delivery or activate alternate metabolic pathways that do not require O2. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza, Hematol. Oncol. Clinics N.erythropoietin (EPO) (reviewed in Semenza, Hematol. Oncol. Clinics N. Amer. 8:863-884, 1994), vascular endothelial growth factor (VEGF) (Shweiki et al., Nature 359:843-845, 1992; Banai et al., Cardiovasc. Res. 28:1176-1179, 1994; Goldberg and Schneider, J. Biol. Chem. 269:4355-4359, 1994), and glycolytic enzymes (Firth et al., Proc. Natl. Acad. Sci. USA 91:6496-6500, 1994; Semenza et al., J. Biol. Chem. 269:23757-23763, 1994).
The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and thus blood O2-carrying capacity (Jelkmann, 1992, supra; Semenza, 1994, supra). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 3xe2x80x2-flanking region and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription. In particular, inducers of EPO expression (1% O2, cobalt chloride [CoCl2], and desferrioxamine [DFX]) also induced HIF-1 DNA binding activity with similar kinetics. In addition, inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity. Furthermore, mutations in the EPO 3xe2x80x2-flanking region that eliminated HIF-1 binding also eliminated enhancer function (Semenza, 1994, supra). These results support a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation participates in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (Semenza, 1994, supra).
EPO expression is cell type specific, but induction of HIF-1 activity by 1% O2, CoCl2, or DFX was detected in many mammalian cell lines (Wang and Semenza, Proc. Natl. Acad. Sci. USA 90:4304-4308, 1993). The EPO enhancer directed hypoxia-inducible transcription of reporter genes transfected into non-EPO-producing cells (Wang and Semenza, 1993, supra; Maxwell et al., Proc. Natl. Acad. Sci. USA 90:2423-2427, 1993). RNAs encoding several glycolytic enzymes were induced by 1% O2, CoCl2, or DFX in EPO-producing Hep3B or nonproducing HeLa cells whereas cycloheximide blocked their induction and glycolytic gene sequences containing HIF-1 binding sites mediated hypoxia-inducible transcription in transfection assays (Firth et al., 1994, supra; Semenza et al., 1994, supra). These experiments support the role of HIF-1 in activating homeostatic responses to hypoxia.
Hypoxia inducible factor-1(HIF-1) is a mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia (Wang, G. L., et al., Proc. Natl. Acad. Sci. USA 92:5510-5514, 1995; Wang, G. L., and Semenza, G. L., J. Biol. Chem. 270:1230-1237, 1995; U.S. Pat. No. 5,882,914). HIF-1 is a basic helix loop-helix protein that binds to cis-acting hypoxia-responsive elements of genes induced by hypoxia (Wang, G. L., and Semenza, G. L., Curr. Opin. Hematol. 3:156-162, 1992; Jiang, B. H., et al., J. Biol. Chem. 212:19253-19260, 1997). The genes that are activated by HIF-1 in cells subjected to hypoxia include EPO, vascular endothelial growth hormone (VEGF), heme oxygenase-1, inducible nitric oxide synthase, and glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1 (Semenza, G. L., et al., Kid. Int. 51:553-555, 1997). HIF-1 DNA binding activity and HIF-1 protein concentration increase exponentially as cells are subjected to decreasing O2 concentrations (Jiang, B. H., et al., Am J. Physiol. 271:C 172-C1180, 1996).
HIF-1 also activates transcription of the VEGF gene in hypoxic cells (Forsythe et al., 1996; Iyer et al., 1998). When cultured cells are transfected with pCEP4/HIF-1alpha plasmid under conditions that allow expression of HIF-1alpha from a cytomegalovirus promoter and a reporter plasmid containing the hypoxia response element from the VEGF gene, reporter gene expression is increased in cells under non-hypoxic conditions and there is a dramatic superinduction under hypoxic conditions that is dependent upon the presence of an intact HIF-1 binding site (Forsythe et al., 1996). In embryonic stem cells from a knockout mouse, which lack HIF-1alpha expression, there is no expression of VEGF mRNA in response to hypoxia (Iyer et al., 1998).
HIF-1 is a heterodimer of two subunits, HIF-1alpha and HIF-1beta. The HIF-1alpha subunit is unique to HIF-1, whereas HIF-1beta (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) can dimerize with other proteins. The concentration of HIF-1alpha and HIF-1beta RNA and HIF-1alpha and HIF-1beta polypeptide increases in cells exposed to hypoxic conditions (Wiener, C. M., et al., Biochem. Biophys. Res. Commun. 225:485-488, 1996; Yu, A. Y., et al., Am J. Physiol. 275:L818-L826, 1998).
Structural analysis of HIF-1alpha revealed that dimerization requires two domains, termed HLH and PAS. DNA binding is mediated by a basic domain (Semenza, G. L., et al., Kid. Int. 51:553-555, 1997). Two transactivation domains are contained in HIF-1alpha, located between amino acids 531 and 826. The minimal transactivation domains are at amino acid residues 531-575 and 786-826 (Jiang, B. H., et al., 1997, supra; Semenza, G. L., et al., 1997, supra). Amino acids 1-390 are required for optimal heterodimerization with HIF1beta (ARNT) and DNA binding. In addition, deletion of the carboxy terminus of HIF-1alpha (amino acids 391-826) decreased the ability of HIF-1 to activate transcription. However, HIF-1alpha (1-390) was expressed at high levels in both hypoxic and non-hypoxic cells in contrast to full-length HIF-1alpha (1-826) which was expressed at much higher levels in hypoxic relative to non-hypoxic cells (Jiang, B.-H., et al., J. Biol. Chem. 271:17771-17778, 1996). Thus, hypoxia has two independent effects on HIF-1alpha activity: (1) hypoxia increases the steady-state levels of HIF-1alpha protein by stabilizing it (i.e. decreasing its degradation); and (2) hypoxia increases the specific transcriptional activity of theprotein (i.e. independent of the protein concentration).
This invention is based on the discovery and isolation of unique variant forms of HIF-1alpha polypeptide that are stable under hypoxic and nonhypoxic conditions. The invention further includes chimeric proteins having HIF-1alpha DNA binding domain and dimerization domains and a heterologous transactivation domain. Given the structural and functional similarities between HIF-1alpha , HIF-2alpha (also known as EPAS 1, HLF, HRF, and MOP2), and HIF-3alpha (see Gu, Y.-Z., et al., Gene Expr. 7:205-213, 1998) it is understood that HIF-1alpha is described for illustrative purposes, but that all these HIFs are included herein.
A stable HIF-1alpha (sHIF-1alpha ) protein of the invention includes the following properties: (1) sHIF-1alpha will contain the basic-helix-loop-helix-PAS domain of HIF-1alpha that mediates dimerization with HIF-1beta (ARNT) and binding to HIF-1 recognition sites on DNA, e.g., the sequence 5xe2x80x2-TACGTGCT-3xe2x80x2 from the human EPO gene (which was used to purify HIF-1 originally) or the sequence 5xe2x80x2-TACGTGGG-3xe2x80x2 from the human VEGF gene (Forsythe et al., 1996; Semenza and Wang, Mol. Cell. Biol. 12:5447-5454, 1992); (2) sHIF-1alpha will contain deletions or amino acid substitutions that substantially increase its half-life in cells under non-hypoxic conditions such that the sHIF-1alpha protein accumulates to much higher levels than the wild-type HIF-1alpha protein under these conditions. There are many different deletions and/or amino acid substitutions that will result in this effect; multiple examples are provided but these are not limiting; (3) sHIF-1alpha contains one or more transcriptional activation domains derived either from HIF-1alpha or another eukaryotic or viral transcription factor. Depending on the activation domain utilized, the transcriptional activity of sHIF-1alpha may be regulated by oxygen concentration or may be constitutively active regardless of oxygen concentration. sHIF-1alpha mediates increased transcription of hypoxia-inducible genes normally regulated by HIF-1.
In one embodiment, the invention includes an isolated nucleic acid sequence encoding a stable HF-1alpha protein that is a chimeric transactivator. This chimeric transactivator includes: a) a nucleotide sequence encoding a DNA binding domain and a dimerization domain of a hypoxia inducible factor (e.g., HIF-1alpha, HIF-2alpha, or HIF-3alpha); and b) a nucleotide sequence encoding a transcriptional activation domain. The preferred hypoxia inducible factor of the invention is HIF-1alpha.
In another embodiment, the invention provides non-chimeric stable HIF-1alpha polypeptides. Such polypeptides include, but are not limited to, HIF-1alpha amino acid residues 1-391 and 521-826 of SEQ ID NO:1; amino acid residues 1-391 and 549-826 of SEQ ID NO:1; amino acid residues 1-391 and 576-826 of SEQ ID NO:1; amino acid residues 1-391 and 429-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine; amino acid residues 1-391 and 469-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine; amino acid residues 1-391 and 494-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine; amino acid residues 1-391 and 508-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine; amino acid residues 1-391 and 512-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine; and amino acid residues 1-391 and 517-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine.
The invention further provides a method for providing constitutive expression of a hypoxia inducible factor in a cell, under hypoxic or non-hypoxic conditions. The method includes contacting the cell with a nucleic acid sequence encoding a chimeric transactivator protein as described herein, or a stable HIF-1alpha as described herein, under conditions that allow expression of the nucleic acid sequence, thereby providing constitutive expression of a hypoxia inducible factor.
The invention also provides a method for increasing expression of a hypoxia inducible gene in a cell. The method includes contacting the cell with an expression vector containing a polynucleotide encoding a stable HIF-1alpha of the invention or a chimeric transactivator protein as described herein under conditions that allow expression of the nucleic acid sequence contained in the vector thereby providing for increased expression of hypoxia inducible genes in the cell. Such genes include, for example, VEGF.
Further included in the invention is a method for reducing hypoxia or ischemia-related tissue damage in a subject having or at risk of having such damage. The method includes administering to the subject a therapeutically effective amount of a nucleic acid sequence encoding a chimeric transactivator protein as described herein, or a stable HIF-1alpha as described herein, in a pharmaceutically acceptable carrier, thereby inducing gene expression that will reduce, or prevent, or repair tissue damage. Examples of gene products whose expression is induced by sHIF-1alpha resulting in a therapeutic effect include VEGF and other mediators of angiogenesis and insulin-like growth factor 2 (IGF-2) and other factors promoting cell survival (Iyer et al., 1998; Feldser, D., et al., Cancer Res. 59:3915, 1999).
In another embodiment, the invention provides a method for providing prophylactic therapy for tissue in a subject in need thereof comprising administering to the subject an amount of a polypeptide encoded by a polynucleotide encoding a chimeric transactivator protein as described herein, or a stable HIF-1alpha as described herein, such that angiogenesis is induced at levels that are greater than before administration of the polypeptide, thereby providing prophylactic therapy.
In one embodiment, the invention provides a substantially purified polypeptide having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid. Isolated polynucleotides encoding such a polypeptide as well as antibodies which preferentially bind this polypeptide are also provided in a particular embodiment, serine 551 is changed to glycine and threonine 552 to alanine.
In one embodiment, a method is provided for treating a hypoxia-related tissue damage in a subject, by administering to the subject a therapeutically effective amount of a nucleotide sequence comprising an expression control sequence operatively linked to a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid.
In another embodiment, the invention provides a method of treating a hypoxia-related tissue damage in a subject by administering to the subject a therapeutically effective amount of a polypeptide having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid.
In a further embodiment, the invention provides a formulation for administration of stable human hypoxia inducible factor-1 (HIF-1alpha) polypeptide to a patient having hypoxia related tissue damage. The method includes a substantially pure polypeptide having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid; and a pharmaceutically acceptable carrier.
The invention also provides a formulation for administration of a polynucleotide encoding stable human hypoxia inducible factor-1 (HIF-1alpha ) to a patient having hypoxia related tissue damage, including a therapeutically effective amount of a nucleic acid sequence comprising an expression control sequence operatively linked to a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid; and a pharmaceutically acceptable carrier.